BackgroundDramatic changes in gene expression occur in response to extracellular stimuli and during differentiation. Although transcriptional effects are important, alterations in mRNA decay also play a major role in achieving rapid and massive changes in mRNA abundance. Moreover, just as transcription factor activity varies between different cell types, the factors influencing mRNA decay are also cell-type specific.Principal FindingsWe have established the rates of decay for over 7000 transcripts expressed in mouse C2C12 myoblasts. We found that GU-rich (GRE) and AU-rich (ARE) elements are over-represented in the 3′UTRs of short-lived mRNAs and that these mRNAs tend to encode factors involved in cell cycle and transcription regulation. Stabilizing elements were also identified. By comparing mRNA decay rates in C2C12 cells with those previously measured for pluripotent and differentiating embryonic stem (ES) cells, we identified several groups of transcripts that exhibit cell-type specific decay rates. Further, whereas in C2C12 cells the impact of GREs on mRNA decay appears to be greater than that of AREs, AREs are more significant in ES cells, supporting the idea that cis elements make a cell-specific contribution to mRNA stability. GREs are recognized by CUGBP1, an RNA-binding protein and instability factor whose function is affected in several neuromuscular diseases. We therefore utilized RNA immunoprecipitation followed by microarray (RIP-Chip) to identify CUGBP1-associated transcripts. These mRNAs also showed dramatic enrichment of GREs in their 3′UTRs and encode proteins linked with cell cycle, and intracellular transport. Interestingly several CUGBP1 substrate mRNAs, including those encoding the myogenic transcription factors Myod1 and Myog, are also bound by the stabilizing factor HuR in C2C12 cells. Finally, we show that several CUGBP1-associated mRNAs containing 3′UTR GREs, including Myod1, are stabilized in cells depleted of CUGBP1, consistent with the role of CUGBP1 as a destabilizing factor.ConclusionsTaken together, our results systematically establish cis-acting determinants of mRNA decay rates in C2C12 myoblast cells and demonstrate that CUGBP1 associates with GREs to regulate decay of a wide range of mRNAs including several that are critical for muscle development.

Small RNA cloning and sequencing is uniquely positioned as a genome-wide approach to quantify miRNAs with single-nucleotide resolution. However, significant biases introduced by RNA ligation in current protocols lead to inaccurate miRNA quantification by 1000-fold. Here we report an RNA cloning method that achieves over 95% efficiency for both 5′ and 3′ ligations. It achieves accurate quantification of synthetic miRNAs with less than two-fold deviation from the anticipated value and over a dynamic range of four orders of magnitude. Taken together, this high-efficiency RNA cloning method permits accurate genome-wide miRNA profiling from total RNAs.

Type I myotonic dystrophy (DM1) is caused by a triplet repeat expansion in the 3-untranslated region (UTR) of the dystrophia myotonia protein kinase (DMPK) gene. Pathogenesis is closely linked with production of a toxic RNA from the mutant allele, which interferes with function of several RNA-binding proteins, including CUGBP1. Here we show that expression of a mutant DMPK 3-UTR containing 960 CUG repeats is sufficient to increase expression and stability of an mRNA encoding the potent proinflammatory cytokine, tumor necrosis factor (TNF). CUGBP1 specifically recognizes sequences within the TNF 3-UTR that are dissimilar from its canonical UG-rich binding site. Depletion of CUGBP1 from mouse myoblasts results in increased abundance of TNF mRNA through stabilization of the transcript. Moreover, activation of the protein kinase C pathway by treatment with phorbol ester, which has been shown previously to result in CUGBP1 phosphorylation, also causes TNF mRNA stabilization. Our results suggest that the elevated serum TNF seen in DM1 patients may be derived from muscle where it is induced by expression of toxic DMPK RNA. Importantly, overexpression of this potent cytokine could contribute to the muscle wasting and insulin resistance that are characteristic of this debilitating disease. Myotonic dystrophy type I (DM1)3 is a debilitating autosomal dominant disorder caused by a triplet CTG repeat expansion in the 3Ј-untranslated region (UTR) of the dystrophin myotonia protein kinase (DMPK) gene (1-3). The disease is characterized by myotonia, progressive muscle wasting, insulin resistance, and cardiac conduction defects (4). Although some of the symptoms of DM1 may be attributed to reduced levels of the DMPK protein, much of the disease pathogenesis is recapitulated by expression of the mutant DMPK 3Ј-UTR (5, 6), or even by elevated expression of the wild type DMPK 3Ј-UTR (7), in otherwise normal mice. This is, at least in part, because transcripts containing the expanded CUG repeat accumulate in nuclear foci where they sequester cellular proteins (8).The function of two RNA-binding proteins, CUGBP1 (CUGbinding protein 1) and MBNL (Muscleblind), is profoundly affected in DM1 patient cells. MBNL associates with the repeatcontaining mRNA and is confined to nuclear foci (8), whereas CUGBP1 is overexpressed and hyperphosphorylated in the nucleus (9, 10). There is also evidence that CUGBP1 may be depleted from the cytoplasm (10). Interestingly, CUGBP1 and MBNL are both splicing regulators that play antagonistic roles in splice site selection. Consistent with this, several clinically relevant mRNAs, including the muscle-specific chloride channel (11), the insulin receptor (12), and cardiac troponin T (13), exhibit aberrant splice patterns in DM1 patient cells.Several studies have indicated that aberrant function of CUGBP1 is a key factor in development of DM1 (5, 6, 14), and it is therefore important to consider other cellular functions of this protein that may be affected. It has been known for some time that CUGBP1 regulate...

PARN is one of several deadenylase enzymes present in mammalian cells, and as such the contribution it makes to the regulation of gene expression is unclear. To address this, we performed global mRNA expression and half-life analysis on mouse myoblasts depleted of PARN. PARN knockdown resulted in the stabilization of 40 mRNAs, including that encoding the mRNA decay factor ZFP36L2. Additional experiments demonstrated that PARN knockdown induced an increase in Zfp36l2 poly(A) tail length as well as increased translation. The elements responsible for PARN-dependent regulation lie within the 3′ UTR of the mRNA. Surprisingly, changes in mRNA stability showed an inverse correlation with mRNA abundance; stabilized transcripts showed either no change or a decrease in mRNA abundance. Moreover, we found that stabilized mRNAs had reduced accumulation of pre–mRNA, consistent with lower transcription rates. This presents compelling evidence for the coupling of mRNA decay and transcription to buffer mRNA abundances. Although PARN knockdown altered decay of relatively few mRNAs, there was a much larger effect on global gene expression. Many of the mRNAs whose abundance was reduced by PARN knockdown encode factors required for cell migration and adhesion. The biological relevance of this observation was demonstrated by the fact that PARN KD cells migrate faster in wound-healing assays. Collectively, these data indicate that PARN modulates decay of a defined set of mRNAs in mammalian cells and implicate this deadenylase in coordinating control of genes required for cell movement.

✉ XPO5 mediates nuclear export of miRNA precursors in a RanGTP-dependent manner. However, XPO5-associated RNA species have not been determined globally and it is unclear whether XPO5 has any additional functions other than nuclear export. Here we show XPO5 pervasively binds to double-stranded RNA regions found in some clustered primary miRNA precursors and many cellular RNAs. Surprisingly, the binding of XPO5 to pri-miRNAs such as mir-17~92 and mir-15b~16-2 and highly structured RNAs such as vault RNAs is RanGTPindependent. Importantly, XPO5 enhances the processing efficiency of pri-mir-19a and mir-15b~16-2 by the DROSHA/DGCR8 microprocessor. Genetic deletion of XPO5 compromises the biogenesis of most miRNAs and leads to severe defects during mouse embryonic development and skin morphogenesis. This study reveals an unexpected function of XPO5 for recognizing and facilitating the nuclear cleavage of clustered pri-miRNAs, identifies numerous cellular RNAs bound by XPO5, and demonstrates physiological functions of XPO5 in mouse development.